Pallis Monica, Burrows Francis, Whittall Abigail, Boddy Nicholas, Seedhouse Claire, Russell Nigel
BMC Pharmacol Toxicol. 2013 Jun 15;14:32. doi: 10.1186/2050-6511-14-32.
Dormant cells are characterised by low RNA synthesis. In contrast, cancer cells can be addicted to high RNA synthesis, including synthesis of survival molecules. We hypothesised that dormant cancer cells, already low in RNA, might be sensitive to apoptosis induced by RNA Polymerase II (RP2) inhibitors that further reduce RNA synthesis.
We cultured leukaemia cells continuously in vitro in the presence of an mTOR inhibitor to model dormancy. Apoptosis, damage, RNA content and reducing capacity were evaluated. We treated dormancy-enriched cells for 48 hours with the nucleoside analogues ara-C, 5-azacytidine and clofarabine, the topoisomerase targeting agents daunorubicin, etoposide and irinotecan and three multikinase inhibitors with activity against RP2 - flavopiridol, roscovitine and TG02, and we measured growth inhibition and apoptosis. We describe use of the parameter 2 × IC50 to measure residual cell targeting. RNA synthesis was measured with 5-ethynyl uridine. Drug-induced apoptosis was measured flow cytometrically in primary cells from patients with acute myeloid leukaemia using a CD34/CD71/annexinV gating strategy to identify dormant apoptotic cells.
Culture of the KG1a cell line continuously in the presence of an mTOR inhibitor induced features of dormancy including low RNA content, low metabolism and low basal ROS formation in the absence of a DNA damage response or apoptosis. All agents were more effective against the unmanipulated than the dormancy-enriched cells, emphasising the chemoresistant nature of dormant cells. However, the percentage of cell reduction by RP2 inhibitors at 2 × IC50 was significantly greater than that of other agents. RP2 inhibitors strongly inhibited RNA synthesis compared with other drugs. We also showed that RP2 inhibitors induce apoptosis in proliferating and dormancy-enriched KG1a cells and in the CD71neg CD34pos subset of primary acute myeloid leukaemia cells.
We suggest that RP2 inhibitors may be a useful class of agent for targeting dormant leukaemia cells.
休眠细胞的特征是RNA合成水平低。相比之下，癌细胞可能依赖于高水平的RNA合成，包括存活分子的合成。我们推测，RNA已经很低的休眠癌细胞可能对RNA聚合酶II（RP2）抑制剂诱导的凋亡敏感，因为这些抑制剂会进一步降低RNA合成。
我们在mTOR抑制剂存在的情况下体外连续培养白血病细胞以模拟休眠状态。评估凋亡、损伤、RNA含量和还原能力。我们用核苷类似物阿糖胞苷、5-氮杂胞苷和氯法拉滨、靶向拓扑异构酶的药物柔红霉素、依托泊苷和伊立替康以及三种对RP2有活性的多激酶抑制剂——黄酮哌啶醇、罗斯考维汀和TG02处理富集休眠细胞48小时，并测量生长抑制和凋亡情况。我们描述了使用2×IC50参数来测量残留细胞靶向性。用5-乙炔基尿苷测量RNA合成。使用CD34/CD71/膜联蛋白V门控策略在急性髓性白血病患者的原代细胞中通过流式细胞术测量药物诱导的凋亡，以识别休眠凋亡细胞。
在mTOR抑制剂存在的情况下连续培养KG1a细胞系诱导出休眠特征，包括RNA含量低、代谢低以及在无DNA损伤反应或凋亡的情况下基础ROS形成低。所有药物对未处理的细胞比对富集休眠的细胞更有效，这突出了休眠细胞的化疗抗性本质。然而，RP2抑制剂在2×IC50时的细胞减少百分比显著高于其他药物。与其他药物相比，RP2抑制剂强烈抑制RNA合成。我们还表明，RP2抑制剂可诱导增殖的和富集休眠的KG1a细胞以及原代急性髓性白血病细胞的CD71neg CD34pos亚群发生凋亡。
我们认为RP2抑制剂可能是一类用于靶向休眠白血病细胞的有用药物。
